JPH0619346B2 - Analytical element for hydrogen peroxide determination - Google Patents

Description

TECHNICAL FIELD The present invention relates to an analytical element useful for quantifying hydrogen peroxide or a substance that produces hydrogen peroxide in the presence of a substance having a peroxidation effect.

BACKGROUND OF THE INVENTION As a method for measuring glucose, cholesterol, uric acid, etc., generally, a target substance is quantified by quantifying hydrogen peroxide produced by the action of its oxidase such as glucose oxidase, cholesterol oxidase, uricase, etc. Method is used.

As a method for quantifying these hydrogen peroxides, colorimetric determination is performed by changing a chromogen to an oxidative type in the presence of a substance having a peroxidation effect, or by oxidizing and condensing one or two chromogens. Well known how to do. Among them, for example, in a dry analytical element, in the presence of a substance having a peroxidative effect,
A diffusion-resistant pyrazolone compound, a diffusion-resistant acylacetamide compound, a diffusion-resistant phenol compound or a diffusion-resistant naphthol compound, and an aromatic primary amine compound or a salt thereof which forms a dye by a coupling reaction with these compounds. A method of quantifying hydrogen peroxide or a substance that produces hydrogen peroxide is disclosed in JP-A-57-94653 and 57-9.
4654, 57-94655, 57-94656, etc.

The method of using a combination of compounds as described above as a chromogen is useful because it has relatively high quantitative sensitivity as compared with conventionally known chromogens, but is useful for biological fluid samples (eg, serum).
When low levels of uric acid, creatinine, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), etc. present in hydrogen peroxide are introduced into hydrogen peroxide and quantified, the concentration of hydrogen peroxide to be quantified is extremely high. Since it is very low, it cannot be said that the discrimination sensitivity for these quantitative determinations is sufficient.

[Object of the Invention] An object of the present invention is to provide an analytical element having remarkably excellent quantitative sensitivity in the quantification of hydrogen peroxide or a substance that produces hydrogen peroxide in the presence of a substance having a peroxidation action. It is in.

[Structure of the Invention] The above object of the present invention is to provide a substance having a peroxide action, a compound represented by the following general formula [I], [II] or [III],
And hydrogen peroxide containing an aromatic primary amine compound or a salt thereof which is oxidized to form a dye by a coupling reaction with a compound represented by the following general formula [I], [II] or [III] Achieved by a quantitative analytical element.

General formula [I] General formula (II) General formula (III) In the formula, X represents a hydrogen atom or a group capable of splitting off upon reaction with an oxidized product of an aromatic primary amine compound. Also, R ₁
To R ₄ each represent a hydrogen atom or a substituent.

[Specific Structure of the Invention] In the compound represented by the above general formula [I], [II] or [III] according to the present invention, the substituents represented by R _{1 to} R ₄ are not particularly limited, Examples thereof include groups such as alkyl, aryl, anilino, acylamino, sulfonamide, alkylthio, arylthio, alkenyl, and cycloalkyl. In addition to these, a halogen atom and cycloalkenyl, alkynyl, heterocycle, sulfonyl, sulfinyl, phosphonyl, etc. , Acyl, carbamoyl, sulfamoyl, cyano, alkoxy, aryloxy, heterocycleoxy, siloxy, acyloxy, carbamoyloxy, amino, alkylamino, imide, ureido,
Sulfamoylamino, alkoxycarbonylamino,
Also included are aryloxycarbonylamino, alkoxycarbonyl, aryloxycarbonyl, heterocyclic thio groups, spiro compound residues, bridged hydrocarbon compound residues, and the like.

The alkyl group represented by R _{1 to} R ₄ has 1 to 1 carbon atoms.
It is preferably 32 and may be linear or branched.

The aryl group represented by R _{1 to} R ₄ is preferably a phenyl group.

Examples of the acylamino group represented by R _{1 to} R ₄ include an alkylcarbonylamino group and an arylcarbonylamino group.

Examples of the sulfonamide group represented by R _{1 to} R ₄ include an alkylsulfonylamino group and an arylsulfonylamino group.

Examples of the alkyl component and the aryl component in the alkylthio group and the arylthio group represented by R _{1 to} R ₄ include the alkyl group and the aryl group represented by R above.

The alkenyl group represented by R _{1 to} R ₄ has 2 carbon atoms.
To 32, and a cycloalkyl group having 3 to 1 carbon atoms
2, especially 5 to 7, are preferable, and the alkenyl group may be linear or branched.

The cycloalkenyl group represented by R _{1 to} R ₄ is preferably one having 3 to 12 carbon atoms, particularly 5 to 7 carbon atoms.

The sulfonyl group represented by R _{1 to} R ₄ is an alkylsulfonyl group, an arylsulfonyl group, etc .; The sulfinyl group is an alkylsulfinyl group, an arylsulfinyl group, etc .; The phosphonyl group is an alkylphosphonyl group, an alkoxyphosphonyl group, an aryloxy group. Phosphonyl group, arylphosphonyl group, etc .; Acyl group as alkylcarbonyl group, arylcarbonyl group, etc .; Carbamoyl group as alkylcarbamoyl group, arylcarbamoyl group, etc .; As sulfamoyl group, alkylsulfamoyl group,
Arylsulfamoyl group and the like; as the acyloxy group, an alkylcarbonyloxy group,
Arylcarbonyloxy group etc .; Carbamoyloxy group as alkylcarbamoyloxy group, arylcarbamoyloxy group etc .; Ureido group as alkylureido group, arylureido group etc .; Sulfamoylamino group as alkylsulfamoylamino group, aryl Sulfamoylamino group, etc .; The heterocyclic group is preferably a 5- to 7-membered one, specifically, 2-furyl group, 2-thienyl group, 2-pyrimidinyl group, 2-benzothiazolyl group, etc .; heterocyclic oxy group Is preferably a 5- to 7-membered heterocycle, for example, 3,4,5,6-tetrahydropyranyl-2-oxy group, 1-phenyltetrazole-5-
Oxy group and the like; The heterocyclic thio group is preferably a 5- to 7-membered heterocyclic thio group, for example, 2-pyridylthio group, 2-benzothiazolylthio group, 2,4-diphenoxy-1,3,5-triazole. -6-thio group, etc .; siloxy group, trimethylsiloxy group, triethylsiloxy group, dimethylbutylsiloxy group, etc .; imide group, succinimide group, 3-heptadecylsuccinimide group, phthalimide group, glutarimide group, etc. A spiro compound residue is spiro [3.3] heptane-
1-yl, etc .; as the bridged hydrocarbon compound residue, bicyclo [2.2.
1] heptan-1-yl, tricyclo [3.3.1.1]
^3.7 ] decan-1-yl, 7,7-dimethyl-bicyclo [2.2.1] heptan-1-yl and the like.

Examples of the group capable of splitting off upon reaction with the oxidized product of the aromatic primary amine compound represented by X include a halogen atom (chlorine atom, bromine atom, fluorine atom, etc.) and alkoxy, aryloxy, heterocyclic oxy, acyloxy, Sulfonyloxy, alkoxycarbonyloxy, aryloxycarbonyl, alkyl oxalyloxy, alkoxy oxalyloxy, alkylthio, arylthio, heterocyclic thio, alkyloxythiocarbonylthio, acylamino, sulfonamide, nitrogen-containing heterocyclic ring bonded by N atom,
Alkyloxycarbonylamino, aryloxycarbonylamino, carboxyl, (R ₁ ′ has the same meaning as R _{1 to} R ₄ above, Z ′ represents a group of non-metal atoms necessary for forming a nitrogen-containing heterocycle, and Z ′ is
The ring formed by may have a substituent. R ₂ ′ and R ₃ ′ represent a hydrogen atom, an aryl group, an alkyl group or a heterocyclic group. ) And the like, but preferably a halogen atom, particularly a chlorine atom.

The nitrogen-containing heterocycle formed by Z'includes a pyrazole ring, an imidazole ring, a triazole ring, a tetrazole ring and the like, and as the substituent which the ring may have, the above-mentioned R _{1 to} R ₄ may be mentioned. There are some.

Among the compounds represented by the above general formulas [I] to [III], particularly preferred are the compounds represented by the general formula [I].

The general formula [I], [II] or [III] according to the present invention will be described below.
Representative specific examples of the compound represented by are shown below, but the present invention is not limited thereto.

1-1 1-2 1-3 1-4 1-5 1-6 1-7 1-8 1-9 1-10 1-11 1-12 1-13 1-14 1-15 1-16 1-17 1-18 1-19 1-20 1-21 1-22 1-23 1-24 1-25 1-26 1-27 1-28 1-29 1-30 1-31 1-32 1-33 1-34 1-35 1-36 1-37 1-38 1-39 1-40 1-41 1-42 1-43 1-44 1-45 1-46 1-47 1-48 1-49 1-50 1-51 1-52 1-53 1-54 1-55 1-56 1-57 1-58 1-59 1-60 1-61 1-62 1-63 1-64 1-65 1-66 1-67 In addition to the above representative examples, specific examples of the compound represented by the general formula [I], [II] or [III] according to the present invention include Nos. 66 to 122 in Japanese Patent Application No. 61-9791. Among the compounds listed on the page, mention may be made of compounds not mentioned herein.

Also, the compounds are described in Journal of the Chemical Society, Perkin I (1977), 2047-2052, US Pat.
3,725,067, JP-A-59-99437, 58-42045, 59-
No. 162548, No. 59-171956, No. 60-33552, No. 60-43659
Nos. 60-172982 and 60-190779, etc. can be referred to for synthesis.

The aromatic primary amine compound according to the present invention includes known compounds widely used as a color developing agent in various color photographic processes, for example, o-
Or a p-aminophenol type compound and an o- or p-phenylenediamine type compound are mentioned. Preferably,
It is an o- or p-phenylenediamine compound, and a p-phenylenediamine compound is particularly preferable.

In the present invention, the preferable p-phenylenediamine compound is represented by the following general formula [IV].

General formula [IV] In the formula, A and B ′ each represent a hydrogen atom or an alkyl group, and A and B ′ may form a heterocycle together with a nitrogen atom. D ', E, G and J each represent a hydrogen atom, a halogen atom, a hydroxy group, an amino group, an alkoxy group, an acylamide group, an arylsulfonamide group, an alkylsulfonamide group or an alkyl group.

As the alkyl group represented by A and B ′, those having 1 to 6 carbon atoms are preferable, and those having 1 to 4 carbon atoms are particularly preferable. These alkyl groups include those having a substituent,
Examples of the substituent include an ureido group, a tetrahydrofurfuryl group, a carboxyl group, a methanesulfonamide group,
Examples thereof include a sulfo group, a methoxy group, an ethoxy group, a methoxyethoxy group, a methoxyethoxyethoxy group, and a methoxytetraethoxy group.

D ', G and J are preferably a hydrogen atom, an alkoxy group, an alkylsulfonamide group and an arylsulfonamide group, more preferably a hydrogen atom. E is preferably a hydrogen atom, an alkyl group or an acylamido group, more preferably an alkyl group having 1 to 3 carbon atoms, and these alkyl groups are the same as the above-mentioned substituents of the alkyl group represented by A and B '. It may have a substituent. The compound represented by the general formula [IV] is generally used in the form of a salt because it is more stable than the free state. Examples of these salts include hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, sulfonic acid, sulfinic acid, sulfuric acid ester, sulfamic acid, thiosulfuric acid S-ester, carboxylic acid, phosphoric acid, phosphoric acid ester, amidophosphoric acid and phosphorous acid. Mention may be made of salts of inorganic or organic acids such as acid esters and organic boron compounds, with hydrochlorides, sulfates and p-toluenesulfonates being particularly preferred.

Typical examples of the aromatic primary amine compound according to the present invention are shown below, but the present invention is not limited thereto.

2-1 2-2 2-3 2-4 2-5 2-6 2-7 2-8 2-9 2-10 2-11 2-12 2-13 2-14 2-15 2-16 2-17 2-18 2-19 2-20 Various substances can be used as the substance having a peroxidizing action according to the present invention, and a typical example thereof is peroxidase. Peroxidase is an enzyme that catalyzes a reaction when hydrogen peroxide oxidizes another substance. This peroxidase is a complex protein that generally contains iron porphyrin, and is present in horseradish, potato, fig sap, turnip (plant peroxidase), milk (lactoperoxidase) and white blood cells (veldoperoxidase), and also in microorganisms. Is also present and can be obtained by extraction or fermentation.

In addition, "Acta Chemica Second Scandinavian, (Acta Chem.
Secand.) Vol. 4, pp. 422-434, 1950, by Theorell and Maehly ", synthetic peroxidases can also be used in the present invention. In addition to peroxidase, methemoglobin, oxyhemoglobin,
Hemoglobin, alkaline hematin, hemin, hemin derivatives and the like can also be used in the present invention.

Other than the enzyme, for example, iron thiocyanate, iron stannate, ferrous ferrocyanate, and a second chromium salt (eg potassium chromium sulfate) adsorbed on silica gel can be used as a substance exhibiting a peroxidation action.

Of these, peroxidase is preferred.

The analysis element of the present invention refers to a dry type element in which reagents necessary for analysis are incorporated in a dry state. As these analysis elements, a single-layered one using a filter paper, a membrane filter or the like as the material of the layer supporting the reagent (Japanese Patent Publication No. 36-4198, U.S. Pat. No. 3607093, etc.),
A glass filter or a membrane filter is used as a non-body type multi-layer analytical element which is simply laminated or peeled off (Japanese Patent Laid-Open No. 49-11395), and two filter papers are covered with a net (Japanese Patent Laid-Open No. 54-54). -151096, US Pat. No. 3,526,480) and the like.

Further, as the analytical element of the present invention, preferably, an integrated multi-layer analytical element having at least one reagent layer and a porous spreading layer on a liquid-impermeable, light-permeable support (Japanese Patent Publication No. 53-2).
1677, JP-A-55-164359, 55-90859, 57-19
7466, 57-101760, 57-101761, 58-90167, etc.).

The reagent layer can be provided as a layer by coating a water-soluble polymer or a hydrophilic and organic solvent-soluble polymer as a binder on a support. Examples of the water-soluble polymer binder include gelatin, gelatin derivatives such as phthalated gelatin, water-soluble cellulose derivatives such as hydroxyethyl cellulose and carboxymethyl cellulose sodium salt, polyvinyl alcohol, poly (N-vinylpyrrolidone), polyacrylamide, polymethacrylamide, and acrylamide. Acrylic ester copolymer, poly (mono or dialkyl substituted) acrylamide,
Examples thereof include poly (mono- or dialkyl-substituted) methacrylamide and water-soluble copolymers thereof, and gelatin, polyacrylamide, and copolymers of acrylamide and acrylic acid ester are preferably used. As the hydrophilic and organic solvent-soluble polymer binder, poly (N-vinylpyrrolidone), poly (N-vinylimidazole),
Examples thereof include poly (N-vinyltriazole) and derivatives thereof or copolymers thereof, and cellulose derivatives such as ethyl cellulose and methyl cellulose. These polymer binders are hydrophilic polymeric substances which are mainly soluble in alcohols such as ethanol, propanol and butanol.

The polymer binder can be arbitrarily selected according to the selected specific component and its analysis reaction. Further, when the selected analysis reaction is composed of two or more kinds of reagents, even if the reagents are mixed and contained in the same reagent layer, two or more kinds of the two or more reagents are contained. It may be contained as a separate reagent layer. These may be determined by the mechanism of action of the analytical reaction itself, and the constitution thereof is arbitrary as long as it does not have an unfavorable influence.

The thickness of the reagent layer can be arbitrarily selected as desired, but it is preferably 1 to 200 μm, more preferably 5 to 100 μm.

The porous spreading layer has (1) a function of uniformly distributing a fixed volume of fluid sample to the reagent layer per unit area. Furthermore, the performance described in JP-B-53-21677, that is, (2) a function of removing a substance or a factor that inhibits an analytical reaction in a fluid sample and / or (3) spectroscopic advanced analysis is performed. It is preferable that it has a function of performing a back ground function of reflecting measurement light that sometimes passes through the support. Therefore, the porous spreading layer according to the present invention is either a layer having only the function of (1) above, or a layer having the functions of (2) and / or (3) in addition to (1). Alternatively, it is possible to appropriately separate a plurality of functions including (1) and use a different layer for each function. Further, of the functions (1), (2) and (3), a layer having two functions and a layer having the remaining one function can be used in combination. For example, a development layer of a non-fibrous porous medium called a brush polymer composed of titanium dioxide and cellulose diacetate described in JP-B-53-21677, described in JP-A-55-164356. Development layer of woven hydrophilically treated fabric, JP-A-57-94658, 57-12847, 57-197466 and 58-
Examples thereof include a fiber structure developing layer described in each specification such as 70161, and a particle-bound structure developing layer described in JP-A-58-90167. In particular, the above-mentioned fibrous structure-developed layer and particle-conjugated structure-developed layer are particularly useful as a material capable of rapidly transferring a blood cell portion. The film thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, but it is preferably about 100 to 600 μm, more preferably about 150 to 400 μm. The porosity is preferably about 20 to 85%.

Depending on the selected specific component and its analysis reaction, the porous spreading layer contains a reagent that directly or indirectly participates in the specific component in the fluid sample, as in the case of the reagent layer described above. You can

As other additional additives, various additives such as preservatives and surfactants can be added as desired.

In particular, the surfactant can be effectively used for controlling the permeation rate when the fluid sample is applied to the analytical element of the present invention.

As the usable surfactant, ionic (anionic or cationic) and nonionic surfactants can be used, but nonionic surfactants are effective. Examples of nonionic surfactants include, for example, 2,5-di-
Examples thereof include polyalkylene glycol derivatives of alkyl-substituted phenols such as t-butylphenoxy polyethylene glycol, p-octylphenoxy polyethylene glycol, p-isononylphenoxy polyethylene glycol, and higher fatty acid polyalkylene glycol esters. These surfactants have the effect of controlling the permeation rate of the fluid sample into the reagent layer and, at the same time, suppressing the undesirable occurrence of “chromatographic phenomenon”.

The surfactant may be used in a widely selected amount, but the amount is from 25% by weight to 0.005% by weight based on the weight of the coating solution.
%, Preferably 15% to 0.05% can be used.

The liquid-impermeable light-transmitting support (hereinafter, abbreviated as the support according to the present invention) may be of any type as long as it is liquid-impermeable and light-transmitting, for example, cellulose acetate. , As well as various polymeric materials such as polyethylene terephthalate, polycarbonate or polystyrene,
Inorganic materials such as glass can also be used. The thickness of the support according to the present invention is arbitrary, but preferably 5
~ 250 μm. Further, one side surface of the support according to the present invention on the observation side can be optionally processed according to its purpose. Further, a light-transmitting undercoat layer may be used on the side of the support on which the reagent layer is to be laminated to improve the adhesion between the reagent and the support.

The above-mentioned integral multi-layer analysis element is, if necessary, for example, a reflective layer described in U.S. Pat.No. 3,992,158, an undercoat layer, a radiation blocking layer described in U.S. Pat.No. 4,042,335, and U.S. Pat.No. 4,066,403. Barrier layer,
The present invention by arbitrarily combining the migration prevention layer described in US Pat. No. 4,166,093, the scavenger layer described in JP-A-55-90859, and the destructible pod-shaped member described in US Pat. No. 4,110,079. The configuration may be arbitrary according to the purpose of.

Various layers of these analytical elements are used by appropriately selecting a slide hopper coating method, an extrusion coating method, a dip coating method or the like conventionally used in the photographic industry according to a desired structure on the support according to the present invention. By sequentially laminating the layers, a layer having an arbitrary thickness can be applied.

The method of adding the compound represented by the general [I], [II] or [III] of the present invention to the liquid for forming the analytical element according to the present invention is, depending on the chemical structure of the above compound,
It can be appropriately selected. For example, various methods such as a method of dissolving in water, an aqueous alkali solution, an aqueous buffer solution, an organic solvent or the like, a solid dispersion method, a latex dispersion method, an oil-in-water emulsion dispersion method, and the like can be used.

The oil-in-water emulsion dispersion method can be a method of dispersing a hydrophobic additive such as a coupler conventionally known in the photographic industry, and is usually dissolved in a high boiling solvent and / or a constant boiling solvent,
It is mixed with an aqueous solution containing a hydrophilic colloid such as gelatin containing an anionic surfactant and / or a nonionic surfactant, and then emulsified and dispersed by a high speed rotation mixer, colloid mill, flow jet mixer, ultrasonic dispersion device, etc. be able to.

As the high boiling point solvent, for example, organic acid amides, carbamates, esters, ketones, urea derivatives and the like, in particular,
Di-n-butyl phthalate, tricresyl phosphate, triphenyl phosphate, diisooctyl acetate, di-n-butyl sebacate, tri-n-hexyl phosphate, N, N-diethylcaprylamidobutyl, N, N-diethyllauryl Amide, n-pentadecyl phenyl ether, dioctyl phthalate, n-nonyl phenol, 3-pentadecyl phenyl ethyl ether, 2,5-di-sec-amyl phenyl butyl ether, monophenyl-di-o-chlorophenyl phosphate, fluorine paraffin, etc. Is mentioned. Among these, dialkyl phthalates, especially 1 to 6 carbon atoms
Those having an alkyl group of are preferred.

Examples of the low boiling point solvent include methyl acetate, propyl acetate, butyl acetate, butyl propionate, cyclohexanol, diethylene glycol monoacetate, nitromethane, carbon tetrachloride, chloroform, cyclohexane, tetrahydrofuran, methyl alcohol, acetonitrile, N, N-dimethyl. Formamide, dioxane, methyl ethyl ketone and the like can be mentioned.

Examples of the anionic surfactant include alkylbenzene sulfonic acid, alkylnaphthalene sulfonic acid, and the like.
Examples of nonionic surfactants include sorbitan sesquioleate, sorbitan monolaurate, and the like.

The method for adding the aromatic primary amine compound or salt thereof according to the present invention to the liquid for forming the analytical element according to the present invention is a method of adding it by dissolving it in water, an aqueous buffer solution, an organic solvent or a solid. Various methods such as a dispersion method can be used.

The analytical element of the present invention is a substance having the above-mentioned peroxide action,
In addition to the compound represented by the general formula [I], [II] or [III] and the aromatic primary amine compound or a salt thereof, an oxidase which produces hydrogen peroxide depending on the kind of the component to be analyzed, and Various reagents such as other enzymes, substrates, buffers, preservatives, surfactants, hardeners can be contained.

Examples of the oxidase that produces hydrogen peroxide include glucose oxidase, uricase, cholesterol oxidase, glycerol oxidase, and glycerin-3.
-Phosphate oxidase, sarcosine oxidase, pyruvate oxidase, D-aspartate oxidase, D (or L) -amino acid oxidase, L-gulono-γ-lactone oxidase, L-sorbose oxidase, L-2-hydroxy acid Oxidase, 6-hydroxy-D-nicotine oxidase, 6-hydroxy-L
- nicotine oxidase, pyridoxamine-phosphate oxidase, pyridoxine oxidase, hexose oxidase, o- aminophenol oxidase, amine oxidase (pyridoxal-containing, or flavin-containing), xanthine oxidase, alcohol oxidase, ethanolamine oxidase, N ⁶ - methyl -L
-Lysine oxidase, choline oxidase, acyl C
Specific examples include oA oxidase and sulfite oxidase.

The substance having a peroxidative effect according to the present invention can be used in a widely selected amount, but in the case of peroxidase, for example, 100 to 1,000,000 U / m ² , preferably 1,
It can be used in the range of 0000 to 100,000 U / m ² .

The compound represented by the general formula [I], [II] or [III] according to the present invention can be used in a widely selected amount, but 0.1 to 100 mmol / m ² , preferably 0.5 to
It can be used in the range of 50 mmol / m ² .

The aromatic primary amine compound or salt thereof according to the present invention may be used in a widely selected amount of 0.005
~ 100 mmol / m ² , preferably 0.01-50 mmol / m
It can be used in the range of ² .

In the analytical element of the present invention, hydrogen peroxide or hydrogen peroxide produced by the action of an oxidase oxidizes the aromatic primary amine compound according to the present invention by the action of a substance having a peroxidation action. The resulting oxidized product of the aromatic primary amine compound is coupled with the compound represented by the general formula [I], [II] or [III] according to the present invention to form a dye. The dye produced is 500-600 nm
It has a visible absorption mainly in and develops color with extremely high sensitivity. Therefore, in the analysis element of the present invention, the analyte is, for example, a trace component in human serum (eg, uric acid, creatinine, GOT, G
It also reacts sensitively to (PT, etc.) and is particularly useful for quantifying trace components.

In the present invention, the substance having a peroxide action according to the present invention, the compound represented by the general formula [I], [II] or [III] and the aromatic primary amine compound can be used, for example, in an integrated multilayer analysis element. In this case, it may be contained in any of the reagent layer, the porous spreading layer and the other layers, but preferably the compound represented by the general formula [I], [II] or [III] and the aromatic first group The primary amine compounds are separated and contained in separate layers. For example, a substance having a peroxidation effect and a compound represented by the general formula [I], [II] or [III] are contained in one reagent layer, and an aromatic primary amine compound is contained in another reagent layer or a porous layer. The embodiment in which it is contained in the development layer is preferable.

When quantifying hydrogen peroxide or a substance that produces hydrogen peroxide using the analytical element of the present invention, the analytical element is immersed in a fluid sample that is a specimen, or a fluid sample is dropped on the analytical element and reflected. It can be measured by spectrophotometry according to the initial velocity method or the reaction end point method. The measured value thus obtained can be applied to a calibration curve prepared in advance to determine the amount of hydrogen peroxide or the substance that produces hydrogen peroxide.

The fluid sample applied to the analysis element of the present invention may be a biological or non-biological fluid sample as long as it contains hydrogen peroxide or a substance that produces hydrogen peroxide. For example, blood (including plasma / serum), lymph, urine, etc. may be mentioned.

In the case of a test piece, the amount of the fluid sample to be used is arbitrary as long as it is equal to or larger than the amount by which the absorbent carrier containing the reagent is sufficiently impregnated with the fluid sample. On the other hand, although it is optional in the case of an integrated multi-layer analytical element, it is preferably about 50 μl to about 5 μl, more preferably about 20 μl to about 5 μl. Usually about 10
It is preferred to apply μl of fluid sample.

[Examples] Hereinafter, the present invention will be described more specifically with reference to Examples.

Example 1 (Analytical element for uric acid) A reagent layer having the composition shown in Table 1, an intermediate layer having the composition shown below, and a developing layer were sequentially provided on a transparent subbed polyethylene terephthalate support having a thickness of 180 μm, and Table 2 was used. The analytical elements 1 to 5 and the comparative analytical element-1 of the present invention shown in FIG.

Comparative compound (1) * 1) Compounds 1-4, 1-5, 1-8, 1-2 of the present invention
All of 0, 1-34 and Comparative Compound (1) were dissolved in ethyl acetate and dibutyl phthalate, added to an aqueous solution of sodium triisopropylnaphthalene sulfonate and gelatin, and ultrasonically dispersed before use.

* 2) Ascorbate oxidase uses cucumber origin.

Intermediate layer (I-1) N-vinylpyrrolidone-vinyl acetate copolymer (weight ratio 2: 8) 1.25 g / m ² Development layer (S-1) Fiber for filter paper raw material [Toyo Roshi Kaisha, Ltd. 40-100 mesh] ] 91.0 g / m ² styrene-glycidyl methacrylate 23.0 g / m ² copolymer (weight ratio 9: 1) Triton X-100 9.1 g / m ² Compound 2-16 (p-toluenesulfonate) of the present invention
0.11 g / m ² dimedone 1.35 g / m ² ascorbate oxidase 7,500 U / m ² bovine serum albumin 1.10 g / m ² * 3) The development layer is coated with xylene solvent.

Compound 2-16 of the present invention (p-toluenesulfonate)
Dissolve dimedone in methanol, add and disperse, and apply.

* 4) Ascorbic acid oxidase (origin from cucumber) was dissolved in water together with bovine serum albumin, freeze-dried, and then finely pulverized to be applied.

The above-mentioned analytical elements-1 to 5 of the present invention and comparative analytical element-1
On the other hand, 10 μl of various uric acid concentrations were dropped on the spreading layer and incubated at 37 ° C. for 7 minutes, and then the reflection concentration was measured from the support side using a 546 nm filter. Got

As is clear from the results of Table 3 above, the analytical elements-1 to 5 of the present invention show a good color density difference with respect to the difference in uric acid concentration, as compared with the comparative analytical element-1, and the discrimination ability, that is, It can be seen that the quantitative sensitivity is high.

Example-2 (Analytical element for uric acid) A reagent layer having the composition shown in Table-4, an intermediate layer having the composition shown below and a developing layer were sequentially provided on a transparent undercoated polyethylene terephthalate support having a film thickness of 180 µm, and shown in Table-5. The analytical elements-6 to 10 and the comparative analytical element-2 of the present invention shown below were prepared.

Comparative compound (2) * 5) Compounds 1-2, 1-4, 1-51, 1-53, 1-65 of the present invention and comparative compound (2) were all dispersed in the same manner as in the method described in Example-1. use.

Intermediate layer (I-2) N-vinylpyrrolidone-vinyl acetate copolymer (weight ratio 7: 3) 1.25 g / m ² Development layer (S-2) Fiber for filter paper raw material [Toyo Roshi Kaisha, Ltd. 40-100 mesh] ] 91.0 g / m ² Styrene-glycidyl methacrylate 23.0 g / m ² copolymer (weight ratio 9: 1) Triton X-100 9.1 g / m ² Compound 2-20 (sulfate) of the present invention 0.06 g / m ² Dimedone 1.35 g / m ² ascorbate oxidase * 4) 20,000 U / m ² bovine serum albumin * 4) 2.5 g / m ² * 6) The development layer was coated with xylene solvent.

Compound 2-20 (sulfur salt) and dimedone of the present invention are dissolved in methanol, added and dispersed, and applied.

The analytical element of the present invention-6 to 10 and the comparative analytical element-
In the same manner as in Example-1, human serum with various uric acid concentrations was added dropwise to 10 μl of the developing layer in Example 2 and incubated at 37 ° C. for 7 minutes, and then the analysis element of the present invention-6 to
The reflection density was measured from the support side using a 546 nm filter for No. 10 and a 650 nm filter for Comparative Analysis Element-2.

As a result, it was found that the analytical elements-6 to 10 of the present invention showed a better difference in color density with respect to the difference in uric acid concentration than the comparative analytical element-2, and the discrimination ability, that is, the quantitative sensitivity was high.

Example-3 (Analytical element for total cholesterol) A reagent layer having a composition shown in Table-6, an intermediate layer or a reagent layer having a composition shown in Table-7, and a table were formed on a transparent undercoated polyethylene terephthalate support having a film thickness of 180 µm. A development layer having the composition shown in FIG.
15 and Comparative Analysis Elements-3 and 4 were prepared.

To the analytical elements-11 to 15 and the comparative analytical elements-3 and 4 of the present invention, 10 μl of human serum having various total cholesterol concentrations was dropped on the spreading layer and incubated at 37 ° C. for 7 minutes, and then 546 nm. The reflection density was measured from the support side using the filter of No. 3, and the results shown in Table 10 were obtained.

As is clear from the results shown in Table-10 above, the analytical elements-11 to 15 of the present invention are compared with the comparative analytical elements-3 and 4,
It shows that the difference in color density is good with respect to the difference in total cholesterol concentration, and that the discrimination ability, that is, the quantitative sensitivity is high.

Example-4 (Analysis element for glucose) A reagent layer having the composition shown in Table-11, an intermediate layer having the composition shown below, and a developing layer were sequentially provided on a transparent subbed polyethylene terephthalate support having a film thickness of 180 µm, and shown in Table-12. The analytical elements-16 to 20 and comparative analytical element-5 of the present invention shown below were prepared.

Intermediate layer (I-4) N-vinylpyrrolidone-vinyl acetate copolymer (weight ratio 2: 8) 1.40 g / m ² Development layer (S-5) * 3) Fiber for filter paper raw material [Toyo Filter Paper () 40- 100 Messhiyu] 91.0 g / m ² styrene - glycidyl methacrylate copolymer (weight ratio 9: 1) 23.0g / m ² Triton X-100 9.1g / m ² the compounds of the invention 2-16 (p-toluenesulfonate )
0.15g / m ² Dimedone 1.90g / m ² Human serum with various glucose concentrations of 10 μm was added to the above-mentioned analytical elements-16 to 20 of the present invention and comparative analytical element-5.
After being dropped on the 1-developed layer and incubated at 37 ° C. for 7 minutes, the reflection density was measured from the support side using a 546 nm filter.

As a result, it was found that the analytical elements-16 to 20 of the present invention showed a better difference in color density with respect to the difference in glucose concentration, and higher discrimination ability, that is, quantitative sensitivity, than the comparative analytical element-5.

[Effects of the Invention] As described in detail in detail, the analytical element of the present invention has a remarkable effect that the quantitative sensitivity is remarkably improved.

Claims (1)

[Claims] 1. A substance having a peroxidation action, a compound represented by the following general formula [I], [II] or [III], and a compound represented by the following general formula [I], [II] or [III] when oxidized. ] An analytical element for hydrogen peroxide determination, which comprises an aromatic primary amine compound or a salt thereof which forms a dye by a coupling reaction with a compound represented by General formula [I] General formula (II) General formula (III) [In the formula, X represents a hydrogen atom or a group capable of splitting off upon reaction with an oxidized product of an aromatic primary amine compound. Also, R
_{1 to} R ₄ each represent a hydrogen atom or a substituent. ]